Discontinuous Wall Hollow Core Magnet

ABSTRACT

A discontinuous wall magnet having an opening or channel is provided. A bead separation magnet having a discontinuous or segmented wall is also provided. The segmented wall causes bead formation to form in a segmented or gapped ring to allow for easier manual pipetting. Also provided are systems and kits having the inventive magnets. Methods of purifying a macromolecule using the inventive magnets are also provided.

BACKGROUND OF THE INVENTION

Many analytical methods in the field of molecular biological researchoften require the isolation of macromolecules for further study. Forexample, the burgeoning field of genomic research compels efficientisolation and purification of nucleic acids—most commonly DNA andRNA—from a variety of biological samples, such as plasmids, tissuesamples, blood or other bodily fluids, archived samples (FFPE samples),and many others. Nucleic acids can be purified via a variety of methods,including the traditional phenol-chloroform extraction, ethanolextraction, or spin columns. One disadvantage of these methods is theirreliance on centrifugation and/or vacuum steps, which have traditionallybeen hard to automate. Advances in sequencing technology in recentdecades presented a need for automatable methods capable of extractingnucleic acids at high throughput rates. To that end, the use of magneticbeads has emerged as the method of choice because it is simple,inexpensive, efficient, and can be performed manually or by automatedpipettors.

In this technology, microscopic paramagnetic beads are coated withapplication-specific functional groups that allow reversible binding ofeither nucleic acids, proteins, or other macromolecules. To achieve thereversible bond, the biological sample is mixed with a solution of themagnetic beads under chemical conditions that support the affinity ofthe macromolecules to the magnetic bead. This mixture is subsequentlyexposed to a magnetic field, leading to sequestration and immobilizationof the magnetic beads together with the macromolecule of interest.Following this step, the supernatant, which is that portion of thesample fluid that remains after the macromolecule of interest has beenextracted, is removed and discarded. While still immobilized by themagnetic field, the bead-macromolecule complex is washed to furtherremove contaminants. Finally, a buffer is added to the bead complex,which changes the chemical conditions (pH, salt concentration) necessaryto maintain the bond between macromolecules and beads. This change inconditions initiates the elution process, whereby the macromolecules arereleased from the magnetic beads (still immobilized) and are nowfree-floating in the elution buffer in purified form.

The magnetic field used to immobilize the magnetic beads is commonlyprovided by magnets. One example of a magnet used in bead separationtechniques is a standard ring magnet. When standard ring magnets arepositioned below the reaction vessel, the magnetic beads aggregate wherethe magnetic field is strongest and generally form a ring around theperimeter of the vessel bottom, reflecting the shape of the magnet. Thisleaves an area in the center of the ring that is mostly free of beads.Accordingly, in order to aspirate the supernatant, a pipet tip must beinserted into this area at the center of the vessel. When this action isperformed, many times the technician inadvertently or accidentallyaspirates beads along with the supernatant. This task can be verychallenging for some when done manually. The smaller the bead ring, themore difficult to aspirate the supernatant without also accidentallyaspirating the beads. Accordingly, a need exists for a system thatallows for easier aspiration of the solution without inadvertentaspiration of the beads in the vessel when pipetting is performedmanually.

SUMMARY OF THE INVENTION

Macromolecules, such as nucleic acids, can be separated or extracted viaa variety of methods. In one method, complexes are formed betweenmacromolecules and magnetic beads, and the magnetic beads are separatedfrom a mixture, essentially purifying the macromolecules after their“un-complexation” or elution from the beads through changes inconditions. In an embodiment, the complex between the macromolecules andmagnetic beads remains in the vessel aggregating to form of a pattern(e.g., a ring pattern, discontinued ring pattern, or other shapedpattern) and most of the solution is removed, leaving a highconcentration of complex in the vessel.

In an embodiment, the present invention includes a magnet that can beused to isolate/purify macromolecules from a mixture. The mixture, asdefined herein, is any aqueous solution that has at least themacromolecule in addition to the solvent. As an example, it can beextracellular matrix, cell debris, plasma, saliva, etc. Themacromolecules, as defined here, encompass nucleic acids such as DNA orRNA, or proteins such as antibodies. The magnet, in particular, can beused to isolate macromolecules by making them adhere to magnetic beads,after which they can be separated from the mixture. In particular,through changes in the chemical environment macromolecules are made toadhere to the magnetic beads to form a complex. The magnet is then usedto attract the complexes and pull them out of solution. In particular,the magnet of the present invention causes the complex to form anaggregation of bead complexes in a pattern within the vessel. Thesolution can then be removed leaving behind the magnetic beads with themacromolecules adhered thereto.

In an embodiment, the magnet encompassed by the present invention, inone aspect, has a top surface (a first surface) at one end, a bottomsurface (e.g., a second surface) at another end, and an opening (e.g.,tunnel, channel canal, or trough) that extends along the length of themagnet. More specifically, the magnet of the present invention has awall (e.g., cylindrical wall) defining the opening extending from afirst end having a first surface to a second end having a secondsurface. The magnet has one or more discontinuous walls (e.g., one ateither or both ends) wherein at least a portion of the discontinuouswall comprises one or more segments and one or more gaps. Thediscontinuous wall forms a shape configured to form a magnetic field,when in use, within the vessel. The magnet has a side wall, for example,that surrounds the magnet. In an aspect, the wall creates a magneticfield that forms a discontinuous pattern in the vessel such that, whenin use, the complex of macromolecules and paramagnetic beads aggregateand can be separated from the mixture. The discontinuous wall, in anembodiment, has one, two, three or four segments separated by one, two,three or four gaps, respectively, to form a discontinuous shape. Theshape of the wall can form a discontinuous ring, oval, square,rectangular, triangular, diamond, or an irregular shape. The magnet ofthe present invention can be made from one or more pieces.

In another embodiment, a system for isolating macromolecules isdisclosed. In addition to the magnet of the present invention, thesystem can include a vessel for holding a mixture that includes amacromolecule (e.g., DNA). The same types of magnets as encompassed byother embodiments can be included as part of the system as well.

Also disclosed are methods of purifying macromolecules from a liquidsample that contains a mixture. The methods, in an embodiment, includesteps of collecting the liquid in a vessel, adding magnetic beads to thesample, and separating the magnetic bead-macromolecule complex from thesample by placing the vessel in the opening/channel of a magnet. Afterthese steps, the macromolecule can be eluted from the magnetic beads. Inan embodiment, the sample can include an extracellular matrix and themethod may further include a step of lysing the sample before addingmagnetic beads to the sample. The method further includes a step ofpipetting sample manually or using an automated pipette. When pipettingmanually, one can do so at one or more gaps in the wall, which allowsone to access the gap in the bead ring, to avoid accidental aspirationof bead complex. Accordingly, the method further includes manuallypipetting at one or more gaps in the wall, wherein the pipet is insertedinto the vessel at a gap formed by macromolecule-magnetic beadcomplexes. The method allows for aspiration of the supernatant withlittle or no accidental or inadvertent aspiration of the bead complexes,as compared to other magnets that form a continuous ring-shaped band ofbead complexes.

In an embodiment, the present invention includes a kit. The kit cancomprise a magnet, as described herein, and a vessel for holding liquidsamples. Magnetic beads and one or more buffers can also be added aspart of the kit in some embodiments.

Additionally disclosed are magnet plate systems for isolatingmacromolecules. The systems include at least one magnet of the presentinvention, as well as a top plate, a support plate, and a base plate.One or more springs wound around one or more shoulder posts can also beincluded as part of the magnet plate systems. The top plate can includea plurality of magnet receivers, and it can accommodate eithercylindrical shaped magnets or block shaped magnets.

There are many advantages provided by the present invention. The magnetof the present invention allows for easier pipetting because it allowsthe use of the vessel wall as a guide for manual insertion of a pipettewith more room and at a better angle. The magnet of the presentinvention provides a magnetic field that allows a bead pattern to formin the vessel that mirrors the segments in the wall which allows foreasier aspiration of the solution in the vessel without disturbing thebeads. This magnet design allows for easier, more efficient recovery ofmacromolecules.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features, and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like parts are referred to by thesame reference characters across different views. The drawings are notnecessarily to scale, emphasis instead being placed on illustrating theprinciples of the invention.

FIG. 1A is a schematic of the formation of macromolecules andparamagnetic beads created by a ring magnet.

FIG. 1B is a schematic of the formation of macromolecules andparamagnetic beads created by a discontinuous magnet of the presentinvention having two segments and two gaps in its wall, as shown inFIGS. 2A, 3A, and 4A.

FIG. 1C is a schematic of the formation of macromolecules andparamagnetic beads created by a discontinuous magnet of the presentinvention having one segment and one gap in its wall, as shown in FIGS.2B, 3B, and 4B.

FIG. 2A is a schematic of a perspective view of a hollow core magnethaving a discontinuous ring comprising two segments and two gaps.

FIG. 2B and FIG. 2C are schematics of a perspective view of a hollowcore magnet having a discontinuous ring comprising one segment and onegap.

FIG. 3A is a schematic of a top view of a hollow core magnet shown inFIG. 2A having a discontinuous ring comprising two segments and twogaps.

FIG. 3B and FIG. 3C are schematics of a top view of a hollow core magnetshown in FIGS. 2B and 2C having a discontinuous ring comprising onesegment and one gap.

FIG. 4A is a schematic of a side view of a hollow core magnet shown inFIG. 2A having a discontinuous ring comprising two segments and twogaps.

FIG. 4B and FIG. 4C are schematics of a side view of a hollow coremagnet shown in FIGS. 2B and 2C having a discontinuous ring comprisingone segment and one gap.

FIG. 5 is a schematic of a perspective view of a block magnet havingeight individual discontinuous wall magnets integrated therein.

FIG. 6A is a schematic of a top view of a magnet plate having multiplediscontinuous wall magnets that each has a cylindrical shape.

FIG. 6B is a schematic of a perspective view of a magnet plate shown inFIG. 6A having multiple discontinuous wall magnets that each has acylindrical shape.

FIG. 6C is a schematic of a side view of a magnet plate shown in FIG. 6Ahaving multiple discontinuous wall magnets that each has a cylindricalshape.

FIG. 6D is a schematic of a front, profile view of a magnet plate shownin FIG. 6A having multiple discontinuous wall magnets that each has acylindrical shape.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

In many molecular biology procedures, macromolecules are needed in apurified form. For example, to prepare a DNA or RNA sample forsequencing e.g., Next-Generation-Sequencing (NGS), it can be extractedfrom any of a variety of clinical sample types, such as tissue, blood,cheek swabs, sputum, forensic material, FFPE samples etc. For example,in certain NGS procedures, the initial extraction from the primarysample is followed by a multitude of enzymatic reactions called libraryconstruction. Each enzymatic reaction is followed by another extractionstep to isolate conditioned nucleic acid from the reaction mix. Theenzymatic reactions are typically followed by amplification (using PCR)and/or size selection (to limit the distribution of fragment sizes to anarrow band of a few hundred basepairs (e.g. 500-700 bp)). The workflowfrom primary sample to sequencing-ready DNA or RNA may involve from 5-10separate extraction steps. Throughout the workflow, the overall volumeof the mix containing the sample, as well as the sample container canvary significantly; typical volumes range from about 2000 μl to 35 μl.These workflows can be performed manually, or they can be automated toachieve increased throughput and potentially better repeatability.

Depending on the nature of the macromolecule to be extracted as well asthe matrix they are present in, magnetic beads (more precisely:paramagnetic beads) are coated with moieties (e.g., functional groups,other compounds) to which the macromolecules have affinity.Macromolecules include nucleic acids (e.g., DNA, RNA, PNA) and proteins(e.g., antibodies, peptides). Essentially, any macromolecule that can bemade to adhere, reversibly or not, to magnetic beads can be subjected tothe methods disclosed herein. For example, the beads might be coatedwith a carboxylic acid having moiety such as succinic acid. The couplingbetween the beads and the macromolecules might also rely onstreptavidin-biotin or carbo di-imide chemistry. Exemplary coatingsinclude protein A, protein B, specific antibodies, particular fragmentsof specific antibodies, streptavidin, nickel, and glutathione. The beadsthemselves can vary in size, but will have an average diameter (e.g., 1micro-meter). In some embodiments, the paramagnetic properties of thebeads will result from integration of iron into an otherwisenon-magnetic substance (e.g., 4% agarose gel). Magnetic beads, as wellas those that are already coated with various affinity groups, can bepurchased from Sigma-Aldrich Corp. (St. Louis, Mo., USA), LifeTechnologies (Now part of Thermo Fisher Scientific) (Grand Island, N.Y.,USA), Thermo Scientific (Rockford, Ill., USA), EMD-Millipore (Billerica,Mass., USA), New England Biolabs (Ipswich, Mass., USA), GE Healthcare(High Wycombe, UK), and Bangs Laboratories (Indianapolis, Ind.).

In one application of the methods of the present invention, molecules(e.g., macromolecules) can be purified using magnetic beads byperforming the following steps:

-   -   a. mixing the magnetic beads having a particular        affinity-conferring functional group with the sample matrix        containing the molecule of interest in a container (e.g., a        vessel, an Eppendorf tube, a microplate well, a deep well, a PCR        well, round-bottom vessel);    -   b. after the mixing, allowing for specific binding between the        beads and the molecules in conditions suitable therefor (e.g.,        by manipulating the conditions), thus creating bead-molecule        complexes;    -   c. placing the bottom of the vessel on or inside the hollow core        magnet having a discontinuous wall of the present invention;    -   d. allowing the bead-molecule complexes to aggregate (e.g.,        segregate) in a pattern around the inside perimeter of the        vessel (or of each vessel if using multiple ones); and    -   e. removing the supernatant, which would contain unbound,        undesired components;    -   f. performing one or more wash steps by adding a suitable        solvent, e.g., ethanol, followed by removal of the same.

Additional steps can include re-suspending the bead-molecule complexesin a solvent, so as to obtain a solution with a desired volume andconcentration. One can choose the appropriate solvent so that thebinding affinity between the beads and the molecules is decreased,allowing them to dissociate from each other. Or one can repeat the stepsabove to aggregate the magnetic beads again to allow for additionalseparations, depending on the buffer chosen.

Also the beads may be used to either bind the component of interest, forexample nucleic acid molecules, and during the method one discards thesupernatant and elutes the component of interest from the beads.Alternatively, one can let the beads bind to a component that one istrying to discard, leaving only the component of interest in thesupernatant. In this case, the supernatant is transferred to a new,clean vessel for use or further experimentation and the magnetic beadswith their unwanted molecules are discarded.

The above methods can be performed manually or by using automatedrobotic systems (e.g., automated liquid handling workstations) oraspirating/dispensing manifolds. Usable workstations for automationinclude Agilent Bravo, the Beckman Biomek i-series, Eppendorf epMotion,Hamilton Star, Tecan Fluent, and many others. When pipetting manually,the technician must take great care to avoid touching the ring ofmagnetic beads that has formed around the vessel bottom perimeter withthe pipet tip, because such contact may cause a portion of the beads,along with their payload (i.e. the extracted macromolecules), to enterthe pipet tip and subsequently be aspirated into the tip and discardedalong with the supernatant. To avoid touching the beads, the pipet tipneeds to be inserted perfectly straight and dead center into the vessel,which requires skill, practice, and dexterity. This task is simplifiedby the design of the magnet of the present invention having adiscontinuous or segmented wall described herein. When magnetic beadsare brought into the proximity of the magnet (by placing the vessel ontop of the magnet), the beads will aggregate at the location of thehighest magnetic field strength, which is generally at the closestdistance from the magnet. If the magnet has a segmented wall, themagnetic beads will reflect that pattern and aggregate in a similarlysegmented way, leaving one or more gaps in the vessel bottom perimeter.See FIG. 1B and 1C. This gap provides an opportunity for the technicianto slide the pipet tip down along the vessel wall, thus using it as aguide, without disturbing the bead ring, because the pipet tip willslide through the opening in the bead ring that was created by the gapin the segmented magnet wall. This way of pipetting greatly reduces therisk of accidentally disturbing the magnetic beads and the resultingbead loss.

Once a complex is formed between a macromolecule of interest and amagnetic bead (which might be formed via covalent as well asnon-covalent bonds), a magnetic field created by a magnet can beemployed to separate the bead-macromolecule complexes from the mixture(e.g., by forming one or more bands of beads in the vessel in closeproximity to the magnet). After that, the supernatant can be aspirated(e.g., via pipetting) and the complexes washed (e.g., with ethanol) tofurther remove contaminants. In a subsequent step the macromolecules canbe released from the beads, for example by eluting them via changes inthe solution (e.g., buffer composition features such as pH and saltconcentration). The present invention allows for easier recovery of theeluate since the discontinuous wall allows the user to easily access theeluate without disturbing the bead formation pattern.

The magnet of the present invention, in one embodiment, is made from arare-earth metal such as neodymium. A neodymium magnet can have thechemical composition Nd₂Fe₁₄B, where Nd is neodymium, Fe is iron, and Bis boron. In some alternative embodiments, the magnet can also be madefrom samarium (e.g., sintered SmCo₅). The magnet can be covered with aprotective layer, for example a layer of nickel. Alternative coatingsinclude one or multiple layers, such as nickel, copper, zinc, tin,silver, gold, epoxy resin, or any other suitable material. Such coatingshelp, among other things, with preventing rusting of the iron component.In each of these embodiments, the full object is referred to as the“magnet”. The magnet can have a strength grade which for differentembodiments can be, for example, about N35, N38, N40, N42, N45, N48,N50, or N52. Additional magnets with different grades, such as thosewith higher N-numbers (those that may be manufactured in the future) ordifferent temperature ranges (H-grades), are also included among theembodiments of the present invention. The magnets (e.g., neodymiummagnets) can be sintered or bonded. Magnets can be purchased from K&JMagnetics, Inc., Jamison, Pa. For example, the openings and thediscontinuous wall can be molded or machined/drilled after sintering butbefore coating and magnetization.

In an embodiment, the magnet of the present invention can be used in anelectromagnetic arrangement in which the magnet is created by use of astainless steel or other ferromagnetic structure having a coil orsolenoid wrapped around it. The solenoid produces a magnetic field whenan electric current is passed through it. This configuration can be usedto form the magnet and system of the present invention. This arrangementand others known in the art, or developed in the future, can be used tocreate the magnet system of the present invention.

The magnet of the present invention has a discontinuous wall instead ofa continuous ring shape, such that, when in use, the magnetic fieldcauses the magnetic beads to form a pattern that is discontinuous or hasgaps. The discontinuous shape of the wall having one or more gapscorresponds to bead pattern formation having one or more gaps thatprovide an opening and better angle for insertion of a pipette. FIG. 1A,1B and 1C show how and where the paramagnetic beads aggregate, and thisoccurs because the shape of the magnetic field changed based on thediscontinuous ring shape of the magnet. In FIG. 1A, the bead formationwas obtained using a ring magnet with a continuous wall. Theparamagnetic beads form a ring shape that coincides with the shape ofthe wall. In FIG. 1B and 1C, performed with the discontinuous ringmagnets shown in FIGS. 2A and 2B, respectively, the paramagnetic beadsform a discontinuous or gapped shape (e.g., ring shape) that mirrors theshape of the wall of the discontinuous ring. As can be seen by FIGS. 1Band 1C, the discontinuous wall magnet allows for separation of themagnetic beads but is better suited for manual pipetting. Thediscontinuous wall allows for a human hand to insert a pipette into thevessel along the side of the vessel and at an angle through anopening/gap in the bead ring that reflects the gap in the wall (FIGS. 1Band 1C), as compared to inserting direct from above (FIG. 1A).

The location of the macromolecule band impacts the steps of themethodology for separating the macromolecules from the mixture. When thevessel is placed on the magnet, the magnetic beads in the solutionaggregate near the magnet at the place of the highest concentration ofthe magnetic field lines; this is where the magnetic field is generallythe strongest. The shape or pattern of the bead formation mirrors theshape of the upper portion of the wall and the bead formation generallyforms in the bottom of the vessel, near the top of the magnet. The shapeof the wall can be chosen based on the separation needs of the user(e.g., manual pipetting, automated pipetting, size of pipettes, volumeof mixture, etc.). After discarding the supernatant and washing theimmobilized beads with a wash solution, the next step is intended torecover the macromolecules from the beads. This is accomplished byexposing the beads to elution buffer, which will reverse the adherencebetween the macromolecules and the beads. The purified macromoleculesare then present in the elution buffer, which can subsequently beremoved from the vessel by aspiration. To effectively elute themacromolecules from the beads, one can add enough elution buffer tocompletely cover the beads with buffer, so that effective elution cantake place. Because it is desirable to keep the elution volume as smallas possible (to achieve a higher concentration of eluate) while ensuringcomplete coverage of the beads by the elution buffer, the magnet of thepresent invention was designed to aggregate the magnetic beads very lownear the bottom of the vessel, regardless of the vessel shape.

Magnetic fields are often visualized using lines. Magnetic field linesare imaginary, but they are helpful tools that illustrate the shape andoutline of a magnetic field. In such illustrations the lines emanatefrom one pole of the magnet and re-enter the magnet at the other pole,thus forming a closed loop. The relative strength of the magnetic fieldat a given location is shown by varying the density of the lines, withhigher densities depicting stronger magnetic fields. The magnetic fieldis strongest at the magnetic poles. The location of the poles on aparticular magnetic shape is determined during manufacturing, when themagnetic material is magnetized. In the present invention, the directionof the magnetization is perpendicular to the surface(s) with the wall,in other words, along the axis of the wall. In particular, the magnetsdisclosed herein are magnetized through the thickness (i.e., along thecenter axis running between the top surface plane and the bottom surfaceplane). Each opening has a top surface and a bottom surface, and eachsuch side (top surface and bottom surface) has a certain polarity, whichcan be designated as north (N) or south (S). When the magnets having anoverall cylindrical shape are assembled on a guide plate (an example ofwhich is shown in FIG. 6A), they can be arranged in any number ofarrangements including alternating rows, alternating columns,checkerboard arrangement or other pattern. Arrangements of polaritiesare embodied for any top plates that might have a different number ofmagnet receivers to accommodate various size plates (e.g., 6, 24, 96,384 or even 1536 sample wells arranged in a 2:3 ratio rectangularmatrix).

Because the shape of the discontinuous wall magnet of the presentinvention is different than that of a standard ring-magnet with acontinuous wall, the magnetic field lines created are different. In themagnet of the present invention, the magnetic field lines result instronger pull forces at or near the segments of the wall, therebyproviding a gap in the formation of the beads to allow for easieraspiration of the solution.

Specifically, magnets having a discontinuous or segmented wall areuseful for manual pipetting to provide a slot or gap into which a pipetcan be inserted by a person. The slot allows for a person to access theliquid in the vessel at an angle using the segmented wall as a guide andsliding the pipet tip through the gap or slot in the aggregatedparamagnetic beads towards the bottom of the vessel without disturbingthe beads.

Referring to FIG. 2A, 2B and 2C show magnets 120A, 120B and 120C. Themagnets have a discontinuous wall (e.g., one or more segments (103A1,103A2, 103B1 and 103C1) and one or more gaps (101A1, 101A2, 101B1 and101C1)). Accordingly, magnets 120A, 120B and 120C each have a channel orcylindrical opening 105A, 105B and 105C, respectively, that go from topsurfaces 104A, 104B, and 104C, and extend to bottom surface 106A, 106B,and 106C, respectively. The sides of magnets 120A, 120B and 120C haveside wall 102A, 102B, and 102C, respectively, that form a cylindricalshape but for the gaps (101A1, 101A2, 101B1 and 101C1) in the wall.

The shape and thickness of the opening or channel can be continuous orcan vary. In the figures, the cylindrical opening or channel isrelatively constant. However, in an embodiment, the channel can besloped, elliptical, curved or have an irregular shape along its length.For example, a sloped opening that slopes inward to reduce the diameterof the opening as it approaches the center of the magnet can be used toaccommodate the shape of vessels that the opening receives. Thisopening, that travels along the length of the magnet, can be any shape(“V”-like shaped, “U” -like shaped or irregular shape) so long as it canreceive the vessel, as described herein.

The overall structure, for magnet 120A, is cylindrical when the presenceof a discontinuous wall and the cylindrical opening are ignored. Inother words, the volume enclosed inside of the outside wall, bound aboveby the plane of the top surface (e.g., a first surface) at one end(e.g., top plane), and bound below by the plane of the bottom surface(e.g., a second surface) at another end (e.g., bottom plane) iscylinder-shaped. When referring to volumes, the terms top surface andbottom surface are used to mean the plane of the top surface at one endand the plane of the bottom surface at the other end, respectively.

As described above, the sides of magnets 120A, 120B and 120C aresurrounded by side wall 102A, 102B and 102C. In the embodiment shown inFIGS. 2A, 2B, and 2C, both the magnet itself is cylindrical and aportion of the wall is cylindrical-shaped or ring shaped. The openingshave walls that are in part cylindrical-shaped and the wall is adiscontinuous wall having gaps 101A1 and 101A2 and segments 103A1 and103A2 such that it forms a discontinuous ring shape. In an embodiment,the wall can be any shape so long as a portion of the wall isdiscontinuous or segmented (e.g., a discontinuous or segmented ringshape) to form a magnet field that attracts the beads in a discontinuouspattern formation within the vessel. The phrase “discontinuous” or“segmented” is used to refer to at least a portion of the wall that haveone or more segments (e.g., one, two, three or four segments) along withone or more gaps, breaks, slots, recesses or the like (e.g., one, two,three or four gaps, respectively).

In an embodiment, the shape of the walls does not need to be a ringshape or cylindrical shape. The wall of the inventive magnet can have atleast a top portion that has a discontinuous or segmented shape of aring, oval, square, rectangular, triangular, diamond, or has a shapethat is irregular. The wall has a shape that forms a magnetic field,when in use, within the vessel. The magnetic field, based on the shapeof the discontinuous or segmented wall, causes the bead to form in apattern that mirrors the wall shape to allow for separation. In anembodiment, the discontinuous wall of the inventive magnet can have atleast a top portion that has any shape so long as it can receive thevessel and, when in use, the magnetic force emanating from the shapeallows the beads/macromolecule complex to aggregate in a pattern suchthat they can be separated from the mixture.

In FIG. 2A, the wall of discontinuous ring of magnet 120A is formed bytwo segments and two gaps. FIGS. 2A-2C show several variations of thediscontinuous ring. Magnets 120B and 120C shown in FIGS. 2B and 2C haveone segment, segment 103B1 or 103C1, respectively, and one gap, gap101B1 or gap 101E1, respectively. Not shown in the figures, areembodiments having three or four segment/gap series. In each instance,the bead formation mirrors the segments of the discontinuous wall. Inthe case of a magnet with a four segment/gap series, four partialcircular bead formations occur and in the case of magnet 120A twopartial circular bead formations occur (see FIG. 1B), whereas in thecase of 120B or 120C one partial circular bead formation occurs (seeFIG. 1C) which can extend along about half or 180° to about two thirdsor 270° (e.g., about 50%, 55%, 60%, 65%, 66%, 70%, 75%, or 80%) of thecircumference of the wall.

Referring to FIGS. 3A-C, the figure shows the top view of the magnetsshown in FIG. 2A-C, respectively. FIGS. 3A, 3B and 3C show the top viewof hollow core magnets 120A, 120B and 120C of FIGS. 2A, 2B and 2C,respectively. In these figures, cylindrical openings 105A, 105B and 105Care shown along with the discontinuous ring arrangement of the wall thatis made up of segments 103A1 and 103A2 and gaps 101A1 and 101A2, segment103B1 and gap 101B1, and segment 103C1 and gap 101C1 respectively.

FIGS. 4A-C show a side view of the magnets shown in FIG. 2A-C,respectively. FIGS. 4A, 4B and 4C show the side view of magnets 120A,120B and 120C, respectively. In these figures, side walls 102A, 102B and102C and cylindrical openings 105A, 105B and 105C are shown along withthe discontinuous ring arrangement made up of two segments 103A1 and103A2 and two gaps 101A1 and 101A2 (not shown), and a single segmentarrangement such as segment 103B1 (FIG. 4B) and segment 103C1 (FIG. 4C)and gap 101B1 (FIG. 4B) and gap 101C1 (FIG. 4C), respectively.

The magnet of the present invention can be a block magnet having anumber of individual openings (e.g., cylindrical opening) integratedtherein. In such a case, the discontinuous wall is embedded around eachopening, but the overall magnet can be block-shaped, a bar, or a prism(e.g., rectangular-prism shaped), as described herein. Briefly, theoverall block shape (or other shape) can have gaps milled, etched,molded, 3D printed, or otherwise inserted to create the discontinuouswall magnet of the present invention. The block magnet can include aplurality of openings (e.g., cylindrical openings) having discontinuousor segmented walls. With respect to the applications of the magnets, thefocus is on the discontinuous wall surround the opening, as opposed tothe full magnet. For example, both the discontinuous wall magnets andthe block magnet having a number of discontinuous walls around openings,are referred to as discontinuous ring magnets, discontinuous wallmagnets or discontinuous magnets because regardless of the shape of theoverall magnet that has openings/channels with a discontinuous wall.FIG. 5 shows block magnet 120D that is bar magnet having eightcylindrical openings (105D), and eight gaps (101D1) and one segment(103D1) forming individual walls. The segments extend from top surface104D or bottom surface 106D (not shown), and the entire block magnet120D is encased by wall 102D. With respect to the applications of themagnets, the focus is on the discontinuous wall, as opposed to the fullmagnet. Therefore, both the individual magnet (e.g., magnet 120A) andthe block magnet (e.g., magnet 120D) are considered and referred to as adiscontinuous wall magnet because regardless of the shape of the magnet,it has a discontinuous wall. The phrase “discontinuous wall magnet” inthis document refers to magnets that have a discontinuous wall and thewall is being shaped to allow for separation of beads in the vessel.

FIG. 6A shows magnet plate 690, within which there is top plate 92 (alsoreferred to as guide plate) that has 96 magnet receivers (i.e., theholes/openings that receive the magnets not shown in the figure). Themagnet receivers are arranged along 8 rows and 12 columns. Each magnetreceiver receives a magnet (e.g., 120A, 120B, 120C). Springs are placedaround shoulder posts at the corners of the top plate. The shoulderposts, and the springs, pass through top plate 92 and base plate 96. Thesprings allow flexibility in the leveling of the magnets, and thus anyvessels placed in their opening or channel. With the springs, pipettingfrom the vessels can be accomplished more efficiently. In an embodiment,support plate 94 is a metal, and an affinity exists between the supportplate and the magnets. Further underneath, below both the top plate andthe support plate, is base plate 96. The top plate can be fastened tothe base plate by inserting shoulder posts (e.g., bolts) through theshoulder bolt receivers found at the corners of the two plates. In someembodiments, the shoulder bolts and the springs can be on each of thefour corners of the plates, whereas in other embodiments they can be inalternative locations (e.g., along portions of the edges or on some ofthe corners only). The support plate is made from a material that hasaffinity to magnets. It can be made from a metal such as iron, nickel,cobalt, or an alloy of different materials (e.g., stainless steel). In asimilar fashion to FIG. 6B, FIG. 6C and 6D show a perspective view, sideview and front view of magnet plate 690. The magnet plates can utilize aplurality of single magnets or block magnets.

The integrated spring components enable complete liquid removal withouttip occlusion. The springs effectively cushion the wells, and allow theplates (e.g., top plate, support plate) to give way when tips (e.g.,pipette tips) come in contact with a well bottom. This compensates forphysical tolerances between labware and pipettors, each of which canotherwise compromise the precision of supernatant removal (e.g.,aspiration).

In general, the magnets of the present invention, when used forisolating macromolecules, allows easier recovery of the macromolecules,especially when pipetting manually. The magnet of the present invention,as described in the example, provides for better separation of the beadsfrom the mixture. This is accomplished because the design of the magnetprovides for a better angle, a guide, and/or space for accessing thesolution in the vessel. As compared to a standard ring magnet, themagnet of the present invention has about the same recovery but allowsthe user to do so in a more accessible fashion. In an embodiment, ascompared to a hollow core magnet having a continuous wall (e.g., astandard ring magnet), a percent recovery using the magnet of thepresent invention having a discontinuous wall is about the same. Inanother embodiment, the percent recovery increases in a range betweenabout 1% to about 35% (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, or 35%), as compared to the amount recovered using astandard ring/continuous wall magnet.

Standard conditions for forming the macromolecule-bead complex are knownin the art and can be found, for example, in Rohland, et al.,Cost-Effective High-Throughput DNA Sequencing Libraries For MultiplexedTarget Capture, Genome Research 22:939-946 and Supplemental Notes (theentire teachings of which are incorporated herein by reference). Forexample, reagent kits that can be used to form the macromolecule-beadcomplex are commercially available, such as the AMPURE composition fromBeckman Coulter, or such reagents can be made. One example of a solidphase reversible immobilization reagent that can be made and used withthe present invention is a MagNA composition, which is made from:

-   -   Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles        (Hydrophylic), 100 mL (GE Healthcare Product No. 45152105050350;        previously known as 0.1% carboxyl-modified Sera-Mag Magnetic        Speed-beads (FisherSci, cat.#: 09-981-123)    -   18% PEG-8000 (w/v) (e.g. Sigma Aldrich, cat.#: 89510)    -   1M NaCl    -   10 mM Tris-HCl, ph 8.0    -   1 mM EDTA, pH 8.0    -   Optional: 0.05% Tween 20        To form the macromolecule-bead complex, in one embodiment,        0.5×-3× MagNA in an amount ranging from 10 microliters to 400        microliters can be added to the mixture.

Exemplification

A High Efficiency 96 Well Magnetic Particle Separation Device Designedfor Use with Manual Pipettors

A. Overview

The isolation or purification of macromolecules (e.g., DNA, RNA, andproteins) is routinely required prior to their use in a multitude ofapplications. The use of magnetic particles coated with a variety offunctional groups is widely used for these applications. Althoughinitially most commonly used in high throughput workflows in conjunctionwith liquid handling robotics, magnetic particles are increasingly usedin low to moderate throughput workflows due to their ease of use,efficiency, and low cost. In a typical low to moderate throughputworkflow users accomplish liquid transfer steps using multi or singlechannel manual pipettors in conjunction with a 96 well magnetic particleseparator. Efficient separation and recovery of the paramagneticparticles complexed to the desired macromolecule is dependent on anumber of factors; viscosity and volume of the liquids being used, thetype and design of the vessel or labware being employed, and importantlythe design of the magnetic particle separator. For manual users themagnetic plate must employ powerful magnets and collect the magneticparticles in a fashion that minimizes any inadvertent bead loss due tovariations of individual pipetting techniques.

To this end, a novel highly powerful magnetic particle separator, agapped or a slotted ring magnet (“SRM”), was designed and tested. TheSRM used in the experiment had two segments and two gaps, as shown inFIGS. 2A, 3A, 4A, and 6. This magnet collects and concentrates themagnetic particles into opposing regions near the bottom of the labwarewells, as shown in FIG. 1B. The gapped/slotted design allows manualusers greater flexibility in their approach to removing supernatantswith a higher degree of confidence that magnetic particles will not beinadvertently aspirated during any sample processing steps.

The data below for a discontinuous wall magnet or SRM demonstrates theadvantage of its design when using paramagnetic particles complexed tonucleic acid molecules and extracted by using manual pipettors and acommonly used magnetic particle purification chemistry.

B. Methods and Materials

-   1) Assembly of magnetic particle purification chemistry (MPPC).

50 mLs of a solution containing the following components was assembled:

10 gms of 20% PEG-8000 (w/v), Sigma, cat.#: 89510

20 mL of 5M NaCl, Sigma, cat.#: S7653

500 uL of 100× Tris-EDTA Buffer Solution, Sigma T9285-100

Add Sigma Nuclease Free Water PN:W4502, to a final volume of 50 mL.

Mix all components until solution is clear.

Add 1.2 mLs of Sera-Mag Magnetic Carboxylate Modified Particles.

Mix solution again until magnetic particles are evenly dispersed.

-   2) Recovery Testing in 96-well Costar round bottom microplates,    (Corning, Inc., Cat.#: 3795)

(a) A master mixture of 112 uL of lambda DNA (New England BioLabs, PN:N3011S) at 500 ng/uL, 56 uL Bovine Serum Albumin (BSA Solution, Ambion,PN: AM2616) at 50 mg/mL, 2.8 mL of 1× Tris-EDTA diluted in nuclease freewater (100× Tris-EDTA Buffer Solution (Sigma T9285-100 —diluted in SigmaNuclease Free Water PN:W4502, and 5 mL of WPC (see section 1 above) wasassembled and gently mixed.

The mixture was allowed to incubate for 5 minutes at room temperature.300 uL of the master mixture was added to row A of two separate roundbottom microplates using a 1000 uL single channel pipettor (Rainin,Cat.#: 17014382).

One round bottom microplate was placed on the SRM plate with the otherplate being placed on a regular ring magnet plate. Both microplates wereincubated for 7 minutes to allow the magnetic particles to be collected.To mimic inadvertent particle aspiration, wells Al through A6 wereaspirated using a tracking aspiration (removal of liquid as the pipettip is moving downward) with contact to the left side of the wells asthe pipet tip was lowered to the bottom of the well, referred to as theTSW (Tracking Side of Well) method. In wells A7 through A12 the liquidwas aspirated using a tracking aspiration with no contact to the sidesof the wells, referred to as the TDC (Tracking Dead-Center) method.These aspiration procedures were used for all steps in the purificationprocess including ethanol washes and elution. Following supernatantremoval the plates were washed two times with 300 uL 75% ethanol (from amaster mix of 75 mL of Ethanol (98%)(Sigma, cat.#E7023) and 25 mL ofnuclease free water (Sigma, cat.#W4502).

Washing is performed in the following manner: Remove microplate from themagnetic separator; add ethanol, resuspend beads, incubate for 30seconds, place microplate on magnetic separator, and wait for the beadsto collect before removing supernatant.

Following the final ethanol wash, the particles were allowed to dry withthe microplates on the magnetic plates for 7 minutes. Lambda DNA waseluted in 50 uL of 1× Tris-EDTA (as prepared above). DNA concentrationin the eluted samples was measured using a DeNovix Model DS-11 Seriesspectrophotometer. Data was recorded in Table 1 below.

C. Data and Analysis

TABLE 1 Recovery of lambda DNA from round bottom plates - SRM andregular ring magnet plate. Regular Ring Magnet Plate SRM Lambda LambdaDNA Aspiration Well DNA Conc. Aspiration Well Conc. Method Location(ng/uL) Method Location (ng/uL) TSW* A1 35.2 TSW* A1 26.2 TSW* A2 35.6TSW* A2 28.1 TSW* A3 34.7 TSW* A3 24.2 TSW* A4 34.8 TSW* A4 22.5 TSW* A535.1 TSW* A5 26.5 TSW* A6 34.9 TSW* A6 24.0 TDC** A7 34.8 TDC** A7 35.0TDC** A8 36.1 TDC** A8 34.6 TDC** A9 35.4 TDC** A9 34.8 TDC** A10 35.7TDC** A10 35.9 TDC** A11 36.0 TDC** A11 36.2 TDC** A12 33.9 TDC** A1236.0 *TSW = Aspirate while tracking down side of well **TDC = Aspiratewhile tracking dead-center to bottom of well without contact with sideof well

1. Analysis

Mean of SRM-TSW Method: 35.1 ng/ul Mean of SRM-TDC Method: 35.3 ng/ulMean of Regular Ring Magnet Plate - TSW 25.3 ng/ul Method: Mean ofRegular Ring Magnet Plate - TDC 35.4 ng/ul Method: % Difference mean ofSRM-TSW method vs mean of regular 28.0 ring TSW method: % Differencemean of SRM-TDC method vs mean of regular 0.99 ring TDC method: STD Devof SRM-TSW Method:  0.3 ng/ul STD Dev of SRM-TDC Method:  0.8 ng/ul STDDev of Regular Ring Mag. - TSW Method:  2.2 ng/ul STD Dev of RegularRing Mag. - TDC Method:  0.7 ng/ul

D. Conclusion

Based on the analysis of the data use of the slotted/discontinuous wallmagnetic plate or SRM resulted in a 28% increase in lambda DNA recoverywhen using the TSW tracking aspiration method, namely using the gap inthe wall along the side of the wall, as compared to the regular ringmagnet plate having a continuous wall with no gaps for the Costar roundbottom plate.

In addition, no significant difference in lambda DNA recovery wasobserved between the slotted/discontinuous wall magnet plate and thecontinuous wall magnet plate using the TDC method toaspirate-while-tracking to bottom of well without contact with the sidesof well, indicating no reduction in performance when using the slottedplate design.

In conclusion, the slotted/discontinuous wall magnet plate designsignificantly mitigates inadvertent loss of magnetic particles due tovariations in pipetting techniques for manual users of magneticparticle-based workflows.

The relevant teachings of all the references, patents and/or patentapplications cited herein are incorporated herein by reference in theirentirety.

The following applications are related to the invention describedherein: U.S. Application No. (not yet assigned), entitled “Solid-CoreMagnet” by Olaf Stelling, filed (on even date herewith); U.S.application Ser. No. 15/497,858, entitled “Solid-Core Ring-Magnet” byOlaf Stelling, filed Apr. 26, 2017; U.S. application Ser. No.14/515,256, entitled “SOLID-CORE RING-MAGNET” by Olaf Stelling, filedOct. 15, 2014. The entire teachings of the above application areincorporated herein by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A magnet for use in isolating macromolecules froma mixture in a vessel when the macromolecules adhere to paramagneticbeads to form a complex, wherein the magnet comprises: a. a cylindricalwall defining a cylindrical opening extending from a first end having afirst surface to a second end having a second surface; and b. one ormore discontinuous walls wherein at least a portion of the discontinuouswall comprises one or more segments and one or more gaps, wherein atleast a portion of the discontinuous wall extends from the cylindricalwall; wherein the discontinuous wall forms a shape configured to form amagnetic field, when in use, within the vessel.
 2. The magnet of claim1, wherein the wall forms a discontinuous pattern in the vessel suchthat, when in use, the complex of macromolecules and paramagnetic beadsaggregate and can be separated from the mixture.
 3. The magnet of claim1, wherein the wall has one, two, three or four segments separated byone, two, three or four gaps, respectively, to form a discontinuousshape.
 4. The magnet of claim 1, wherein at least the portion of thewall is shaped to form a discontinuous ring, oval, square, rectangular,triangular, diamond, or an irregular shape.
 5. The magnet of claim 1,wherein the magnet is made from one or more pieces.
 6. A magnet for usein isolating macromolecules from a mixture in a vessel when themacromolecules adhere to paramagnetic beads to form a complex, whereinthe magnet comprises: a. a wall defining a lengthwise opening extendingfrom a first end having a first surface to a second end having a secondsurface; and b. one or more discontinuous walls wherein at least aportion of the discontinuous wall comprises one or more segments and oneor more gaps, wherein at least a portion of the discontinuous wallextends from the wall; wherein the discontinuous wall forms a shapeconfigured to form a magnetic field, when in use, within the vessel. 7.The magnet of claim 6, wherein the wall forms a discontinuous pattern inthe vessel such that, when in use, the complex of macromolecules andparamagnetic beads aggregate and can be separated from the mixture. 8.The magnet of claim 6, wherein the wall has one, two, three or foursegments separated by one, two, three or four gaps, respectively, toform a discontinuous shape.
 9. The magnet of claim 6, wherein at leastthe portion of the wall is shaped to form a discontinuous ring, oval,square, rectangular, triangular, diamond, or an irregular shape.
 10. Themagnet of claim 6, wherein the magnet is made from one or more pieces.11. A method for purifying a macromolecule from a liquid sample having amixture, the method comprising: a. collecting the liquid sample in avessel; b. adding magnetic beads to the liquid sample, wherein steps “a”and “b” can be performed in any order under conditions to form amacromolecule-magnetic bead complex between the macromolecule and themagnetic bead; c. separating the complex from the sample by placing thevessel on the magnet or in a of a magnet, wherein the magnet comprises:i. a wall defining a lengthwise opening extending from a first endhaving a first surface to a second end having a second surface; and ii.one or more discontinuous walls wherein at least a portion of thediscontinuous wall comprises one or more segments and one or more gaps,wherein at least a portion of the discontinuous wall extends from thewall; wherein the discontinuous wall forms a shape configured to form amagnetic field, when in use, within the vessel.
 12. The method of claim11, wherein the magnet is made from one or more pieces.
 13. The methodof claim 11, comprising the step of pipetting sample manually or usingan automated pipette.
 14. The method of claim 13, wherein the step ofmanually pipetting occurs at one or more gaps in the cavity wall,wherein the pipette is inserted into the vessel at a gap formed bymacromolecule-magnetic bead complexes.
 15. The method of claim 11,further comprising the step of eluting the nucleic acid material fromthe magnetic beads.
 16. The method of claim 11, wherein the samplecomprises an extracellular matrix, cell debris, plasma, saliva, or acombination thereof.
 17. The method of claim 11, further comprising astep of lysing the sample before adding magnetic beads to the sample.18. A kit for use in isolating macromolecules from a mixture in a vesselwhen the macromolecules adhere to paramagnetic beads to form a complex,wherein the kit comprises: a. a magnet that comprises: i. a walldefining a lengthwise opening extending from a first end having a firstsurface to a second end having a second surface; and ii. one or morediscontinuous walls wherein at least a portion of the discontinuous wallcomprises one or more segments and one or more gaps, wherein at least aportion of the discontinuous wall extends from the wall; wherein thediscontinuous wall forms a shape configured to form a magnetic field,when in use, within the vessel; and b. the vessel for holding themixture having the macromolecule, wherein the vessel is placed on themagnet or is shaped to fit within the one or more openings.
 19. The kitof claim 18, wherein the magnet is made from one or more pieces.
 20. Thekit of claim 18, wherein the wall forms a shape configured such that,when in use, the complex of macromolecules and paramagnetic beadsaggregate in a pattern such that they can be separated from the mixture.21. The kit of claim 18, wherein at least the portion of the wall isshaped to form a discontinuous ring, oval, square, rectangular,triangular, diamond, or an irregular shape.
 22. The kit of claim 18,wherein the kit further comprises magnetic beads.
 23. The kit of claim18, wherein the kit further comprises one or more buffer compositions.24. A magnet plate system for use in isolating a macromolecule from amixture in a vessel, wherein the magnet plate comprises: a. at least onemagnet, wherein the magnet comprises: i. a wall defining a lengthwiseopening extending from a first end having a first surface to a secondend having a second surface; and ii. one or more discontinuous wallswherein at least a portion of the discontinuous wall comprises one ormore segments and one or more gaps, wherein at least a portion of thediscontinuous wall extends from the wall; wherein the discontinuous wallforms a shape configured to form a magnetic field, when in use, withinthe vessel; and b. a top plate adapted to receive a plurality ofmagnets, wherein the top plate has one or more post openings forreceiving a top end of a post and a spring; c. a post having a top endand a bottom end; d. a spring for placement at the opening of the topplate and surrounding the post; e. a support plate to support themagnet, wherein an affinity exists between the support plate and themagnet; f. a base plate receives the bottom end of the post and thespring and is placed underneath the base plate when in use.
 25. Themagnet plate system of claim 24, wherein the magnet is made from one ormore pieces.
 26. The magnet plate system of claim 24, wherein the topplate comprises a plurality of magnet openings to receive the magnets.